PROJECT SUMMARY Staphylococcus aureus is the leading cause of bloodstream, lower respiratory tract, skin and soft tissue infections in the United States. Demonstrating the broad tissue range and virulence properties of the pathogen, S. aureus also causes osteomyelitis, septic arthritis, and a spectrum of toxin-mediated entities including staphylococcal toxic shock syndrome, enterotoxin-induced gastrointestinal disease, and life-threatening desquamation caused by a family of epidermolytic toxins. Recent estimates suggest that S. aureus contributes to half a million infections per year in the United States alone, resulting in over 10,000 deaths. The annual economic burden of S. aureus infection reached $14.5 billion in 2003. To date, there is no commercially available vaccine to prevent S. aureus infection, and novel antimicrobial agents that successfully target this organism have been few. In the context of widespread disease that has been met with a paucity of highly effective, durable anti-infective strategies, it is imperative that we obtain a more detailed understanding of the molecular mechanisms of S. aureus pathogenesis. S. aureus alpha-hemolysin (Hla) is a pore-forming cytotoxin encoded in the genome and expressed by almost all S. aureus strains. Hla contributes to the pathogenesis of pneumonia, primary and recurrent skin infection, and sepsis through its interaction with ADAM10, the toxin's eukaryotic receptor. Hla is a premier target of ongoing clinical vaccine and passive immunization studies. The primary goal of this proposal is to develop a comprehensive knowledge of how the Hla-ADAM10 complex injures a diverse array of cells and modulates tissue repair within the context of specific host tissue microenvironments, thereby enhancing our knowledge of disease progression host susceptibility. This knowledge will enable the rational translation of novel anti-toxin therapies to impact human disease. This proposal is based on four fundamental discoveries: 1) Through the use of cell-type specific ADAM10 knockout mice, we have isolated the effects of Hla on individual cells within specific tissues in well-defined disease states. 2) The actions of Hla are not merely a product of toxic pore-formation, but depend on toxin-mediated activation of ADAM10 and pathologic cleavage of native ADAM10 substrates. 3) We have demonstrated that the physiologic and pathologic manifestations of infection are a composite of Hla action on discrete cell populations, integrated in the tissue over time. 4) We have demonstrated that an anti-Hla antibody response is associated with protection against recurrent S. aureus infection in children. Through studies that reveal the precise mechanism by which the Hla-ADAM10 complex results in host cell and tissue injury, coupled with focused analysis of human susceptibility to Hla-mediated disease, we anticipate that these studies will enable refinement of clinical trials targeting Hla, and inform the approach to disease prevention and therapeutic intervention. Simultaneously, these studies will contribute more broadly to our understanding of bacterial pore forming cytotoxins.